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Mutagenesis of the Yeast V‐ATPase Vo subunit d
Author(s) -
Sorrell Sara,
Owegi Margaret,
Bilbo Sarah,
Pappas Donald,
ParraBelky Karlett
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a519-d
Subject(s) - atp hydrolysis , protein subunit , atpase , v atpase , biochemistry , vacuole , proton transport , mutagenesis , mutant , chemistry , atp synthase gamma subunit , transmembrane domain , alanine , cytosol , amino acid , biophysics , biology , enzyme , membrane , cytoplasm , gene
Vacuoles are storage compartment for calcium and the recycling center for proteins in yeast cells. V‐ATPase proton pumps are large complexes at the vacuolar membrane responsible for maintaining an acidic vacuolar environment critical for vacuolar function. V‐ATPases consist of two domains, V1 and Vo. V1 hydrolyses ATP and Vo forms the pore used to transport protons from the cytosol. The Vo subunit d is the only Vo component that is not integral to the membrane, suggesting that subunit d could be involved in structural and functional coupling ATP hydrolysis and proton transport. To study the role of subunit d for V‐ATPase function, we introduced 11 changes in subunit d by site‐directed mutagenesis. Residues F94, H128, D173, D217, D261, D286, E317, E319, W325, E328, and C329 were replaced by alanine one by one and the effect that each amino acid substitution had on V‐ATPase function was analyzed. This study showed two distinct groups of mutants, those which did not affect ATP hydrolysis (F94, H128, D173, D217, D261, D286) and retained significant levels of V‐ATPase function (73–106 % of the wild‐type ATPase activity), and those mutations, clustered at the C‐end of subunit d, which significantly inhibited ATP hydrolysis (E317, E319, W325, E328, and C329) and retained only 10 – 40 % of the wild‐type activity. We are further characterizing these mutants in order to understand the functional significance of this region in subunit d.